Using genetic mouse models combined with time-lapse imaging in live neurons, we identified syntaphilin (SNPH) as a static anchor for axonal mitochondria required for maintaining a large number of axonal mitochondria in a stationary state through an interaction with the microtubule-based cytoskeleton. Deleting snph results in a robust increase of axonal mitochondria in motile pools, while over-expressing SNPH abolishes axonal mitochondrial transport. The snph knockout mouse is an ideal genetic model to examine the impact of enhanced motility of axonal mitochondria on presynaptic function, mitochondrial quality control, and neurodegeneration. Specific Aim 1. Activity-Dependent Regulation of Axonal Mitochondrial Transport Axonal mitochondria are recruited to synaptic terminals in response to neuronal activity. However, the mechanisms underlying activity-dependent regulation of mitochondrial transport are largely unknown. Using genetic models combined with live imaging, we demonstrated that SNPH mediates the activity-dependent immobilization of axonal mitochondria through binding to KIF5 motors. The KIF5-SNPH coupling inhibits the motor ATPase. Neuronal activity further recruits SNPH to axonal mitochondria. This motor-docking interplay is induced by Ca2+ and synaptic activity and is necessary to establish an appropriate balance between motile and stationary axonal mitochondria. Deleting snph abolishes the activity-dependent immobilization of axonal mitochondria. We propose an Engine-Switch and Brake model, in which SNPH acts both as an engine off-switch by sensing Ca2+ and as a brake by anchoring mitochondria to the MT-track. Our study provides new mechanistic insight into the molecular interplay between motor and docking proteins, which arrests axonal mitochondrial transport in response to neuronal activity. Specific Aim 2. Mobile Axonal Mitochondria Contribute to the Variability of Synaptic Strength One of the most notable characteristics of synaptic physiology in the CNS is the wide pulse-to-pulse variability of synaptic strength in response to identical stimulation. A long-standing question is how this variability arises. In hippocampal neurons, approximately one-third of axonal itochondria are highly motile and some dynamically pass through presynaptic boutons. This raises a fundamental question: Can those motile mitochondria contribute to the pulse-to-pulse variability of presynaptic strength? We has solved this puzzle by combining live neuron imaging and electrophysiological analysis of genetic mouse model in which axonal mitochondrial motility was selectively manipulated. Using hippocampal neurons and slices of snph knockout mice, we demonstrate that the motility of axonal mitochondria correlates with presynaptic variability. Enhancing mitochondrial motility increases the pulse-to-pulse variability, while immobilizing mitochondria reduces the variability. Using dual-channel imaging of axonal mitochondria motility and SV release at single-bouton levels using synapto-pHluorin, we further showed that mitochondrial movement, either into or out of presynaptic boutons, significantly influences SV release due to fluctuation of synaptic ATP levels. An anchored mitochondrion provides stable and continuous ATP supply. A motile mitochondrion passing through temporally supplies ATP, thus changing synaptic energy levels and influencing various ATP-dependent synaptic activities. Therefore, the fluctuation of presynaptic ATP levels resulting from axonal mitochondrial transport contributes to the wide variability of presynaptic strength. Our study revealed, for the first time, that the fast movement of axonal mitochondria is one of the primary mechanisms underlying the presynaptic variation in the CNS. Thus, our study brings new insight into the fundamental properties of the CNS to ensure the plasticity and reliability of synaptic transmission. Specific Aim 3. Altered Mitochondrial Transport Coordinates Mitophagy PINK1/Parkin-mediated pathways ensure mitochondrial integrity and function. Parkin translocation to damaged mitochondria induces mitophagy in many non-neuronal cell types. However, evidence showing Parkin-mediated mitophagy in primary mature neurons is controversial. Our study reveals four unique features for Parkin-mediated mitophagy in mature cortical neurons under our culture conditions. (1) Parkin is selectively recruited to depolarized mitochondria, a process occurring much more slowly than in non-neuronal cells. (2) Parkin translocation only occurs in a small portion of neurons. (3) Chronically dissipating &#916;&#968;m with low concentrations of uncoupling reagents induces mitophagy mainly in the soma and proximal regions of processes. (4) Such compartmental restriction is due to altered motility of depolarized mitochondria with reduced anterograde and enhanced retrograde transport, thus reducing anterograde flux of damaged mitochondria into distal processes. We propose a functional interplay model between mitochondrial motility and mitophagy to ensure proper removal of aged and dysfunctional mitochondria from distal processes. This spatial process allows neurons to eliminate dysfunctional mitochondria via the autophagy-lysosomal system in the soma, where mature lysosomes are relatively enriched. In addition, we established a high-quality mature cortical neuron culture system, in which the majority of neurons survive and mitochondria remain highly motile after chronic treatment with low concentrations of mitochondrial uncoupling reagents. Such chronic mitochondrial stress conditions allow detection of the altered mitochondrial transport and mitophagy events, thus better reflecting chronic mitochondria stress under in vivo physiological or pathological conditions. An important puzzle in the field of PD research is why mice lacking PINK1 or parkin bear only subtle phenotypes related to dopaminergic neuronal degeneration or mitochondrial morphology change. This raises the possibility that other mechanisms may compensate for loss of PINK1 or parkin in neurons. Mitochondrial ubiquitin ligase 1 (MUL1) is a mitochondria-targeted E3 protein ligase. By collaborating with Ming Guos (UCLA), we show that MUL1 acts in parallel to the PINK1/parkin pathway to ensure mitochondrial integrity and function, thus maintaining neuronal health. Loss of both MUL1 and parkin aggravates mitochondrial damage and induces degeneration-like phenotypes in mouse cortical neurons. Specific Aim 4. Organismal Aging Affects Neuronal Mitochondrial Maintenance Aging is associated with cognitive decline and increasing risk of neurodegeneration, thus raising a fundamental question: Does organismal aging affect mitochondrial maintenance in neurons? We address this issue by characterizing the effect of organismal aging on neuronal mitochondria using in vivo imaging of C. elegans. The simple and well-characterized nervous system, translucent body, and short lifespan of C. elegans provide significant advantages for examining in vivo changes in mitochondrial maintenance in a single neuron over the animal's adulthood. Our study establishes that age-related changes in neuronal mitochondria are complex: mitochondrial trafficking in the distal mechanosensory neuron process declines progressively with age, while mitochondrial size, density and resistance to oxidative stress undergo three distinct stages: increase in early adulthood, maintained at high levels during mid-adulthood, and decline during late adulthood. In contrast, long-lived daf-2 mutants exhibit delayed age-associated changes in mitochondrial maintenance during adulthood. Our study reveals in vivo evidence showing dynamic changes in neuronal mitochondrial maintenance during organismal aging.